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Discovery and application of improved delivery method of large vectors in human cells

A vector, large-scale technology, applied in the field of gene editing

Active Publication Date: 2020-07-17
鲲石生物科技(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, virus-mediated transfection has the highest efficiency and low cytotoxicity, but when used in research or clinical applications, it will cause huge biosafety and ethical issues.

Method used

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  • Discovery and application of improved delivery method of large vectors in human cells
  • Discovery and application of improved delivery method of large vectors in human cells
  • Discovery and application of improved delivery method of large vectors in human cells

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Experimental program
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Embodiment Construction

[0029] This specific embodiment is only to introduce the concept of the present invention, and is for explanatory and illustrative purposes, and cannot limit the scope of use of the present invention.

[0030] 1. Cell culture and cell lines: HepG2, Huh7, PC3, SH-SY5Y, HEK293, MCF7, HL-60, A549 cancer cell lines are all from the American Type Culture Collection (ATCC). All cell lines are free of mycoplasma when using mycoplasma detection or mycobacterial probe for periodic detection.

[0031] 2. Cells were cultured in T-75 flasks at 37°C and 5% CO2.

[0032] 3. Use a medium supplemented with 1 / 100 penicillin / streptomycin (P / S, Sigma) and 10% fetal bovine serum (Hyclone).

[0033] 4. Huh7, HepG2, A549, HEK293, MCF7 were cultured in Dulbecco's modified Eagle medium (DMEM, Sigma), HL-60 was cultured in RPMI medium 1640 (Sigma), PC3 and SH-SY-5Y were cultured in DMEM: Cultivate in F-12 (1:1) medium (Gibco).

[0034] 5. The cells divide at a fusion speed of about 70-90%, and the culture med...

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Abstract

The invention relates to the technical field of genome engineering, in particular to an improved delivery method of large vectors (6-15 kb) in human cells. The method comprises following steps: addingsmall (1-3 kb) vectors to the large vectors (6-15 kb), equivalently co-transfecting the small (1-3 kb) vectors and the large vectors (6-15 kb) by electroporation. The method is reasonable in structural design, the limitation of existing methods can be overcome, the cell transfection efficiency and the cell survival rate are remarkably improved, and the method for improving cell transfection through the small vectors can be widely applied to clinical biomedicine and industrial biotechnology, and has good application and popularization prospects.

Description

Technical field [0001] The present invention relates to the technical field of gene editing, in particular to the research and development of a new cell transfection method and its application, and in particular to an improved method of large-scale vector transfection in human cells. Background technique [0002] CRISPR is a repetitive sequence in the genome of prokaryotes. It is an immune weapon developed by prokaryotes to resist virus invasion to resist viruses and integrate their genes into the genome of prokaryotes, thus creating the CRISPR-Cas9 system, through which bacteria can Precisely knock out or inhibit virus replication and reproduction in prokaryotes. [0003] The CRISPR-Cas9 technology is similar to the secondary immune response of mammals. When bacteria resist the invasion of viruses or foreign plasmids, they will produce corresponding "memory" to resist the re-invasion of this kind of foreign genetic material. Immunity is achieved by the bacterial CRISPR-Cas9 syste...

Claims

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Application Information

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IPC IPC(8): C12N15/87
CPCC12N15/87
Inventor 克劳迪娅·库特乔纳斯·内斯科乌·桑德加德尹秀山
Owner 鲲石生物科技(深圳)有限公司
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